ISSN 1742-206X

1742-206X(2010)6:12;1-7

www.molecularbiosystems.org Volume 6 | Number 12 | December 2010 | Pages 2341–2576

PAPERKiessling et al.Peptide ligands that use a novel binding site to target both TGF-β receptors

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2392 Mol. BioSyst., 2010, 6, 2392–2402 This journal is c The Royal Society of Chemistry 2010

Peptide ligands that use a novel binding site to target both

TGF-b receptorsw

Lingyin Li,a Brendan P. Orner,za Tao Huang,b Andrew P. Hinckb and

Laura L. Kiessling*a

Received 24th July 2010, Accepted 3rd September 2010

DOI: 10.1039/c0mb00115e

The transforming growth factor beta (TGF-b) signaling pathway plays myriad roles in

development and disease. TGF-b isoforms initiate signaling by organizing their cell surface

receptors TbRI and TbRII. Exploration and exploitation of the versatility of TGF-b signaling

requires an enhanced understanding of structure–function relationships in this pathway. To this

end, small molecule, peptide, and antibody effectors that bind key signaling components would

serve as valuable probes. We focused on the extracellular domain of TbR1 (TbRI-ED) as a target

for effector screening. The observation that TbRI-ED can bind to a TGF-b coreceptor (endoglin)

suggests that the TbRI-ED may have multiple interaction sites. Using phage display, we identified

two peptides LTGKNFPMFHRN (Pep1) and MHRMPSFLPTTL (Pep2) that bind the TbRI-ED

(Kd E 10�5 M). Although our screen focused on TbRI-ED, the hit peptides interact with the

TbRII-ED with similar affinities. The peptide ligands occupy the same binding sites on TbRI and

TbRII, as demonstrated by their ability to compete with each other for receptor binding.

Moreover, neither interferes with TGF-b binding. These results indicate that both TbRI and

TbRII possess hot spots for protein–protein interactions that are distinct from those used by their

known ligand TGF-b. To convert these compounds into high affinity probes, we exploited the

observation that TbRI and TbRII exist as dimers on the cell surface; therefore, we assembled a

multivalent ligand. Specifically, we displayed one of our receptor-binding peptides on a dendrimer

scaffold. We anticipate that the potent multivalent ligand that resulted can be used to probe the

role of receptor assembly in TGF-b function.

Introduction

TGF-b isoforms, TGF-b1, 2, and 3, are disulfide-linked

homodimers with molecular weights of approximately 25 kDa

(Fig. 1A).1–3 TGF-b signaling occurs upon formation of a

quinary complex that consists of TGF-b and two copies each

of the transmembrane Ser/Thr kinase receptors, TbRI and

TbRII.2,4 Signaling complex formation occurs when TGF-b1or TGF-b3 binds with high affinity (Kd E 5–30 pM) to two

copies of TbRII. The resulting TGF-b:TbRII complex then

recruits two copies of TbRI to form a hetero-oligomeric

complex. The TGF-b2 homolog is lacking two key arginine

residues present in TGF-b1 and TGF-b3 that facilitate high

affinity interactions with TbRII;5 therefore, TGF-b2 requires acoreceptor (b-glycan or TbRIII) to assemble a signaling com-

plex. Once TbRI and TbRII are proximal, the cytoplasmic

domain of TbRII catalyzes the phosphorylation of multiple

TbRI threonine and serine residues within a conserved

juxtamembrane GS domain (a 30-amino acid region that

contains a characteristic SGSGSG sequence). GS domain

phosphorylation promotes activation of the adjacent TbRI kinasedomain. The activated enzyme then catalyzes the phospho-

rylation of the receptor-regulated Smad proteins (R-Smad),6

Smad2 and Smad3, with the help of an adaptor protein SARA

(Smad anchor for receptor activation).7 R-Smads are critical

regulators of TGF-b signaling that shuttle between the cyto-

plasm and nucleus. Upon growth factor stimulation, the

phosphorylated Smad2 or Smad3 dissociates from SARA

and binds to the common Smad (co-Smad), Smad4; this

complex undergoes nuclear translocation. Once in the nucleus,

the Smad complex interacts with various DNA binding partners

to activate or repress the expression of hundreds of genes.

TGF-b-induced changes in gene expression elicit a wide

range of cellular responses, including cell adhesion, migration,

extracellular matrix deposition, proliferation, apoptosis, and

differentiation.3,8 Depending on the cellular context, TGF-bcan play essential or deleterious roles in development, immunity,

wound healing, or cancer. For example, the growth factor

controls embryonic stem cell self-renewal as well as important

developmental processes such as the epithelial to mesenchymal

transition.9 Loss of TGF-b signaling is associated with auto-

immunity, which highlights its role in immune suppression.10

TGF-b is crucial for wound healing, but its prolonged

presence causes inflammation and scar formation.11 Another

role for the growth factor is as a strong tumor suppressor, yet

aDepartment of Chemistry and Biochemistry, University of Wisconsin,Madison, WI 53706, USA. E-mail: kiessling@chem.wisc.edu;Fax: +001 608 265-0764; Tel: +001 608 262-054

bDepartment of Biochemistry, University of Texas Health ScienceCenter, San Antonio, TX 78229, USA

w Electronic supplementary information (ESI) available: Five supple-mentary figures and one supplementary table are included. See DOI:10.1039/c0mb00115ez Present address: Division of Chemistry and Biological Chemistry,Nanyang Technological University, Singapore 637616.

PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems

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it is also implicated in the late stage metastasis of many cancer

types.12

Because of the important and myriad roles of TGF-b, itsligands would be valuable tools. They could be used to probe

its diverse cellular functions and facilitate the identification

of potential therapeutics. Hence, TGF-b isoforms and their

receptors are popular targets for small molecule screens

and antibody-based therapeutics.13 Compounds that inhibit

the TbRI kinase domain and the highly related kinase domains

of another two type I receptors, Activin A and Nodal,

have been sought.14 One such compound, the kinase inhibitor

SB-431542, has become a powerful tool for assessing the

involvement of TGF-b signaling in specific biological

processes. TGF-b2 antisense oligonucleotides,15 neutralizing

antibodies16 and peptide ligands for the growth factor17 have

been developed to dissect the roles of each individual TGF-bisoform. These investigations highlight the utility of com-

pounds that act on targets within the TGF-b pathway for

dissecting the function of TGF-b signaling components in

development and disease.

These valuable tools, combined with structures of the

TGF-b:TbRI-ED:TbRII-ED complex determined by X-ray

Fig. 1 (A) Schematic depiction of the TGF-b signaling pathway. TbRII (green), which forms non-covalent homodimers as well as higher order

oligomers,18 binds to the covalently linked TGF-b homodimer (orange). The TGF-b/TbRII complex then recruits two copies of TbRI (purple).

This quinary complex enables the constitutively active TbRII to catalyze the phosphorylation of the serine residues in the juxtamembrane GS

domain of TbRI. Upon GS domain phosphorylation, the adjacent kinase domain catalyzes the phosphorylation and activation of receptor-

regulated Smad proteins (R-Smad), Smad2 and Smad3 (blue) with the help of an adaptor protein SARA (Smad anchor for receptor activation,

light brown). Phosphorylated Smad2 and Smad3 dissociate from SARA and bind to common Smad (co-Smad), Smad4 (teal), and the complex

translocates into the nucleus. Once in the nucleus, the Smads bind to different DNA binding partners to control gene expression. Structures used in

the creation of this figure were determined by X-ray crystallographic analysis and rendered using PyMOL molecular graphics. PDB files used

follow: PDB ID 3KFD19 (for TGF-b1:TbRI-ED:TbRII-ED ternary structure), 2QLU20 (for activin receptor type IIB cytoplasmic domain residues

188–483, which is homolous to TbRII residues 267–592), 1IAS21 (for TbRI cytoplasmic domain residue 171–503), 1DEV22 and 1U7F23

(for unphosphorylated Smad3 bound to the Smad binding-domain of SARA), 1U7F (for phosphorylated Smad3 and Smad3:Smad3:Smad4

trimeric complex). For extracellular and intracellular segments whose structures have not been determined by X-ray crystallography, online

software NetSurfP (http://www.cbs.dtu.dk/services/NetSurfP/) was used to predict secondary structures, and a-helices and transmembrane

domains are represented by cylinders. (B) Structures of the extracellular domains of TbRI (purple, PDB ID 2PJY: C), TbRII (dark green, PDB ID

2PJY: B),24 BMPR-IA (magenta, PDB ID 2GOO: B) and ActRII (light green, PDB ID 2GOO: C)25 indicate these proteins share a common three-

finger toxin fold stabilized by four disulfide bonds.

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crystallography,19,24 have led to insight into the function of

this canonical signaling complex. The growth factor and its

receptors, however, have additional binding partners, and the

roles of these interactions in TGF-b signaling are less explored.

For example, TGF-b isoforms bind to b-glycan and endoglin.

Endoglin is another type III receptor (sharing 71% amino acid

identity in the transmembrane and cytoplasmic domain with

b-glycan) that is highly expressed in proliferating endothelial

cells. The receptors, TbRI and TbRII, also interact with

endoglin through both their extracellular domains and cyto-

plasmic domains.26 Thus, although TbRI and TbRII have

small extracellular domains (B150 residues) and large surface

areas are buried in the TGF-b:TbRI-ED:TbRII-ED ternary

complex,24 they possess other hot spots27 for protein–protein

interactions. We found this feature of the receptors intriguing.

Because phage-display has been used to identify preferred

binding sites for protein–protein interactions,28,29 we envisioned

applying this method to the TbR extracellular domains to

identify novel TbR ligands.

Phage-displayed peptide library screening is a technique

used to identify ligands for protein targets.29,30 Compounds

that disrupt31 or promote protein–protein interactions,32 func-

tion as hormone or growth factor mimetics,33 or serve as

ligands for whole cells34 have been discovered using this

technology. We screened a phage-displayed peptide library

using TbRI-ED as bait to identify TbR ligands. Intriguingly,

this screen yielded peptide ligands that recognize both TbRI-ED

and TbRII-ED, yet do not compete with TGF-b. Thus, ourdata indicate that TbRI and TbRII share a novel binding site

that may serve as a target for probing and modulating TGF-bfunction.

Experimental

Materials

All reagents for phage panning experiments and solution phase

synthesis were purchased from Sigma Aldrich (Milwaukee, WI)

and used without further purification unless specified other-

wise. BSA (albumin, bovine, pH 7.0, biotechnology grade) was

purchased from Research Organics (Cleveland, OH). M13

Ph.D 12 phage display library kits were purchased from

New England Biolabs (Ipswich, MA). The 96-well plates used

for immobilization of targets in phage panning experiments

and ELISA assays were purchased from Nunc Thermo Fisher

Scientific (Rochester, NY). Anti-M13 antibody conjugated

with HRP (horseradish peroxidase) was purchased from GE

healthcare (Piscataway, NJ). The substrate for HRP, 2,20-

azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) or ABTS

was purchased from Invitrogen (Carlsbad, CA). Peptides

were either purchased from Biomatik (Wilmington, DE) or

synthesized at the Peptide Synthesis Facility at the University

of Wisconsin-Madison and purified by HPLC to >80%

purity. The mink lung epithelial (Mv1Lu) cell line stably

transfected with SBE (CAGA)12-Luc reporter gene was a

generous gift from Professor F. M. Hoffmann (University of

Wisconsin-Madison). Fetal bovine serum (FBS) and DMEM

were purchased from Invitrogen. The Bright-Glot Luciferase

Assay System and CellTiter-Glos Luminescent Cell Viability

Assay were purchased from Promega (Madison, WI).

LumiNunct Plates were purchased from Nunc Thermo Fisher

Scientific. Recombinant human BMPR-IA (Gln24-Arg152) Fc

chimera, recombinant human ActRII (Ser25-Pro134) Fc

chimera, recombinant human endoglin-ED (residues 26–586)

and recombinant human b-glycan-ED (residues 21–781) were

purchased from R&D Systems (Minneapolis, MN).

Protein preparation

Recombinant human TGF-b1 was a generous gift from

Professor F. M. Hoffmann (University of Wisconsin-Madison).

Recombinant human TbRI-ED (residues 1–101) and recombi-

nant human TbRII-ED (residues 1–137) were expressed in

E. coli, refolded and purified as described.35

Phage display and phage ELISA

TbRI-ED (residues 1–101) was immobilized in microtiter wells

by incubating 100 mL of a solution of TbRI-ED (residues

1–101, 15 mg, 10 kDa) 4 1C for 12 h. The wells were exposed to

200 mL of blocking buffer, which consists of 2% BSA in

Tris-buffered saline with detergent (TBST: 50 mM Tris,

150 mM NaCl, 0.05% Tween 20, pH 7.4). The M13 Ph.D

phage library (100 mL at 1012 pfu mL�1) was allowed to bind

to TbRI-ED coated wells for 2 h. Unbound and weakly bound

phage particles were removed by washing 6 � 5 min with

200 mL of washing buffer (0.1% Tween 20 in TBST for the

first round and 0.5% Tween 20 in TBST for rounds 2–4).

Phage particles that bound to TbRI-ED were eluted by a

10 min treatment with 100 mL of 0.1 M glycine buffer (pH 2.2);

the samples were neutralized immediately with 10 mL of 2 M

Tris–HCl buffer (pH 8.2). The resulting phage were amplified

after the first and second rounds of panning. The output phage

from the third round were used as the input for the fourth

round without amplification. After four rounds of panning,

24 phage plaques were sequenced and 7 unique clones were

identified.

Phage clones were evaluated for binding to TbRI and TbRII

using a phage-based ELISA. Equimolar concentration of TbRsolutions were used in immobilization experiments, in which

100 mL of TbRI-ED (1.0 mg) or TbRII-ED (residues 1–137,

1.5 mg, 15 kDa) were immobilized in microtiter wells at 4 1C

for 12 h. The wells were subsequently blocked with a solution

of 6% BSA for 2 h. Phage clones at various concentrations in a

solution of 2% BSA in phosphate buffer saline (PBS) with

0.5% Tween 20 were incubated in TbRI-ED, TbRII-ED or

BSA-coated wells for 1 h at room temperature. In the ELISA

based competition assay, phage clones (305 pM for clone

1 and 350 pM for clone 2) and varying concentrations of

Pep1 or Pep2 (in a solution of 2% BSA in PBS with 0.5%

Tween 20) were mixed together and added to immobilized

TbRI-ED and TbRII-ED. After 4� 5 min washes with 200 mLof 0.5% Tween 20 in PBS, phage that bound to these wells

were detected by exposure to with anti-M13 antibody con-

jugated with HRP for 1 h. Followed by incubation with the

substrate ABTS in the presence of H2O2 for 30 min, the

absorbance at 405 nm of each well was measured on an

ELx800 absorbance microplate reader (BioTek).

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Reporter gene assay

Mink lung epithelial (Mv1Lu) cells stably transfected with a

TGF-b-responsive reporter gene SBE (CAGA)12-Luc was

used. The gene construct consists of twelve repeats of a Smad

binding element with a sequence of CAGA (SBE(CAGA)12)

engineered immediately upstream of a gene encoding luciferase.36

The transfected cells were cultured in 10% FBS in DMEM.37

About 4000 cells were plated into 24-well plates and allowed

to attach overnight in the normal cell culture medium. The

medium was switched to a low serum medium (0.2% FBS in

DMEM) 4 h before TGF-b treatment to eliminate the effect of

TGF-b the serum. Cells were treated with TGF-b1, TGF-b1with serial dilutions of the peptides and peptides alone. Non-

treated cells were used as a control. Three replicates were

performed for each condition. After 18–24 h, the medium was

removed and cells were washed once with PBS. Luciferase

production was quantified using a Bright-Glot Luciferase

Assay System. More specifically, cells were lysed by incubating

with 75 mL of Glo Lysis Buffer for 5 min. The cell lysate (25 mL)was transferred to a 96-well white plate (LumiNunct Plate).

Bright-Glot Assay Reagent containing the luciferin substrate

(25 mL) was added to the cell lysate, followed by immediate

quantification using a luminometer plate reader (Perkin Elmer

Victor 3 from MTX lab systems). The luminescence reading

from each well was normalized by the cell number, which was

determined separately using a CellTiter-Glos Luminescent

Cell Viability Assay.

Dendrimer synthesis

Because the region of sequence variability on phage particles is

at the N-terminus of the PIII coat protein, peptides identified

from the screen were coupled to dendrimers through a C-terminal

modification. A cysteine was installed at the C-terminus and

its nucleophilicity was exploited. A 20% methanol solution of

PAMAM dendrimer (generation 3)38 with an ethylenediamine

core presenting 32 surface amino groups (Sigma Aldrich)

was diluted in 1 M HEPES buffer (pH 7). A bifunctional

N-hydroxysuccinimidyl ester (NHS)–PEG8–maleimide linker

(Thermo Scientific) was dissolved in dimethyl sulfoxide

(DMSO) at 200 mg mL�1 and 64 molar equivalents were

added to the dendrimer solution; this mixture was allowed to

react for 16 h. The remaining NHS–PEG8–maleimide was then

removed using a PD-10 size-exclusion column. The cysteine-

extended Pep1 was appended to the reactive dendrimer through

conjugate addition of the thiolate to the maleimide in 1MHEPES

buffer (pH 7). Cysteine was then added to block any remaining

reactive maleimide groups. The final product was dialyzed in

water (Milli-Q) overnight and then subjected to lyophilization.

The resulting dendrimer was characterized by SDS-PAGE and

MALDI (matrix-assisted laser desorption/ionization) mass

spectrometry and has a molecular weight of B35 kDa.

Surface plasmon resonance (SPR) experiments to evaluate

dendrimer binding

HEPES-buffered saline (HBS from Biacore, pH 7.4) at a flow

rate of 5 mL min�1 was used as the running buffer to generate

protein surfaces on a CM5 sensor chip (Biacore). The sensor

chip was preconditioned by injecting two consecutive 10 s

pulses of each of the following solutions in the order listed:

10 mM aqueous HCl, 50 mM aqueous sodium hydroxide,

0.1% SDS, and water. The flow rate was maintained at

100 mL min�1. Three separate flow cells were functionalized with

TbRI-ED (residues 7–91), TbRII-ED (residues 1–137) and

endoglin-ED (residues 26–586). A protein-free flow cell was

generated as a negative control. The flow rate was maintained

at 5 mL min�1 for surface generation. The carboxymethyl

dextran surfaces were activated through an injection of

25 mL of a 1 : 1 aqueous mixture of 1-(3-dimethylaminopropyl)-

3-ethylcarbodiimide hydrochloride (75 mg mL�1) and

N-hydroxysuccinimide (NHS) (11.5 mg mL�1). Protein attach-

ment presumably occurs via coupling of the succinimidyl

ester functionalized flow cells to the protein Lys side chains.

Injections of 50 mL of 20 mg mL�1 protein solutions in 10 mM

NaOAc (pH 5.0) buffer were used for these reactions. An

injection of 50 mL of ethanolamine (1 M in H2O, pH 8.5) was

added to block any remaining succinimidyl esters. Approxi-

mately 3600 RU of TbRI-ED, 1900 RU of TbRII-ED,

7300 RU of endoglin-ED were immobilized. In a separate

flow cell, ethanolamine was coupled directly to the activated

surface to evaluate non-specific interactions. In a separate

experiment, three distinct flow cells were functionalized with

either 1600 RU of b-glycan-ED, 3300 RU of BMPR-IA-ED,

or 4300 RU of ActRII-ED. Dendrimer binding was tested at

concentrations ranging from 2.9 nM–6 mM. Serial dilutions of

dendrimers in HBS buffer were injected (KINJECT) over all

four flow cells for 5 min and allowed to dissociate for 5 min at

a flow rate of 10 mL min�1. Signals from the negative control

surface were subtracted from signals from protein-immobilized

surfaces using BIAevaluation version 4.1 software. The surfaces

were regenerated after each injection to remove the bound

dendrimer. To optimize the regeneration conditions, solutions

of high salt, high pH, or low pH were tested. The optimal

regeneration conditions, which remove bound dendrimer with-

out compromising activities of immobilized proteins, were

determined to be a 30 s pulse of 100 mM HCl at a flow rate

of 100 mL min�1.

Results and discussion

Identification of peptide ligands for TbRI using phage display

The features of protein–protein interfaces render them

challenging targets for ligand identification. In the case of

TGF-b for example, the structure of the TGF-b:TbRI-ED:

TbRII-ED ternary complex indicates that more than 2000 A2

of solvent accessible area on each receptor is buried.24,39 Given

the large sizes of protein interaction interfaces, it is not

surprising that phage panning experiments tend to yield

successful ligands when libraries composed of peptide sequences

longer than 10-residues are employed. We therefore screened a

library of 1011 random, 12-residue peptides displayed on

the N-terminus of the PIII protein of M13 phage against

immobilized TbRI-ED. Because the goal of our study was to

discover novel ligands for TbRI-ED, we recovered bound

phage particles using acid elution rather than competitive

ligand-based elution. After four rounds of panning, 24 clones

were sequenced, and 7 unique phage-borne peptides were

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identified. We evaluated these clones using a phage enzyme-

linked immunosorbent assay (ELISA).40 In this way, we could

identify clones that exhibit specificity for the target over BSA.

Two such clones emerged; they display the peptide sequence

LTGKNFPMFHRN (clone 1) or MHRMPSFLPTTL (clone 2)

(Fig. S1, ESIw). We further characterized these two peptides.

Evaluation of the affinity and specificity of the peptide ligands

using ELISA

We assessed the affinity of phage clone 1 (Fig. 2A) and clone 2

(Fig. 2B) for immobilized TbRI-ED using a phage ELISA.

Both clones bind TbRI-ED with high affinity (apparent

Kd E 10�10 M) (Table S1A, ESIw). We also tested their ability

to interact with TbRII-ED. Although this receptor does not

share obvious sequence homology with TbRI-ED, the active

clones also interact with the TbRII-ED. Indeed, the measured

affinities were similar to those for the target TbRI-ED.

We suspected that these phage clones bind avidly to the receptors

because each phage particle displays an average of 3–5 copies of

the peptide ligand.41 Thus, the Kd values measured represent

their ‘‘apparent affinity’’.42

To evaluate the affinity and specificity of the monovalent

peptides, we synthesized peptide LTGKNFPMFHRN (Pep1)

and MHRMPSFLPTTL (Pep2) as well as the corresponding

N-terminal fluorophore-labeled counterparts. The use of these

materials in direct binding assays, such as ELISA, SPR, or

fluorescence polarization (FP), should require high micro-

molar to millimolar concentrations. These conditions, how-

ever, can result in aggregation, which would interfere with the

mass transfer or fluorescence polarization output of SPR or

FP assays. Moreover, low affinity ligands tend to have fast

rates of dissociation; therefore, their binding is difficult to

observe in assays that require washing steps, such as ELISAs.

Fig. 2 Phage display against TbRI yields peptides that bind TbRI-ED and TbRII-ED indistinguishably. The binding of (A) phage clone 1 and (B)

phage clone 2 to immobilized TbRI-ED and TbRII-ED was assessed using a phage-based ELISA. (C) ELISA-based competition binding assay.

Pep1 derived from phage clone 1 and (D) Pep2 derived from clone 2 were tested for inhibition of phage clone binding to immobilized receptors

(550 pM of clone 1 and 39 pM of clone 2 were used). (E) An assay with Pep1 competing with phage clone 2 (39 pM) for binding to either

immobilized TbRI-ED or TbRII-ED. The IC50 value for Pep1 with phage clone 2 and TbRI-ED is 110 mM; the corresponding value for TbRII-ED

is 156 mM. (F) An assay with Pep2 competing with phage clone 1 (550 pM) for binding to either immobilized TbRI-ED or TbRII-ED. The IC50

value for Pep2 inhibiting phage clone 1 binding to TbRI-ED is 256 mM; the corresponding value for TbRII-ED is 274 mM. Error bars represent the

mean � the standard deviation in (A) to (F).

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Because the phage clones are multivalent, they will bind avidly

rendering their interactions readily monitored. To take

advantage of the phage detection system and avoid the

problems associated with directly measuring synthetic peptide

binding, we carried out competition ELISAs.31 We reasoned

that adding a peptide that interacts selectively with a given

TbR should cause a decrease in phage binding. A key require-

ment for competition is that the observed signal cannot arise

from phage aggregation or nonspecific binding to the plastic

well, but rather from specific interactions with the immobilized

receptors. To minimize non-specific phage binding, we tested

the influence of two different detergents on binding. The

nonionic detergent, Tween 20, is more effective at disrupting

non-specific phage binding and aggregation. Utilization of

0.5% Tween 20 allowed for competition of synthetic peptides

with the phage-borne peptides (Fig. S2A–C, ESIw).After optimization of the assay conditions, we tested

whether Pep1 or Pep2 could block phage clone binding to

either TbRI-ED or TbRII-ED. Phage (at a constant concen-

tration close to their apparent Kd value) were mixed with

increasing concentrations of the corresponding synthetic

peptide in TbR-coated wells. Both peptides exhibited dose-

dependent competition with either phage clone (Fig. 2C, D).

These data indicate that both Pep1 and Pep2 bind to TbRI and

TbRII. We determined the IC50 values of the peptides by

fitting the competition curves and then derived their Kd using

the Cheng–Prusoff equation: Kd = IC50/(1 + [phage]/Kd phage)43

(Table S1B, ESIw). This analysis revealed that the mono-

valent ligands, Pep1 and Pep2 exhibit reasonable affinities

(Kd E 10�5 M) for both TbRI and TbRII. These results

demonstrate that competition ELISAs of this type can be used

to determine the binding affinities of low-affinity phage-derived

peptides. Importantly for our goals, phage panning against

TbRI-ED yielded peptide ligands that bind to TbRI-ED and

TbRII-ED with similar affinities.

Probing the peptide binding sites on TbRI and TbRII

There is no apparent sequence homology between Pep1 and

Pep2, yet both bind to TbRI and TbRII. These results

prompted us to ask whether they share binding sites on TbRI

and TbRII or whether each occupies a unique site on each

receptor. To this end, we carried out a cross competition assay.

Interestingly, Pep2 inhibited not only phage clone 2 but also

clone 1, and Pep1 similarly inhibited phage clone 2. Thus, each

peptide occupies the same binding site on a given receptor

(Fig. 2E and F).

That seemingly unrelated peptides can bind the same site on

each TbR is a finding that has parallels in other systems.

Specifically, phage panning experiments focused on the

vascular endothelial growth factor (VEGF) yielded three

classes of sequences.31 Although no sequence homology is

apparent among these classes, they all compete with each

other for receptor binding. Structures of two of these peptides

in complex with VEGF have been determined by X-ray

crystallography;44 one peptide binds VEGF using side chain

contacts while the other acts through backbone interactions.

These results emphasize two features of phage display screening.

First, ligands can be found that use very different binding

modes to occupy the same site, and second, these ligands tend

to bind at protein–protein interaction sites.

To narrow the pool of potential peptide binding sites on

TbRI-ED, we employed a truncated version of TbRI-ED

(residues 7–91), which lacks structurally disordered segments

on the N- and C-termini. The affinities for both clones for the

truncated TbRI-ED were similar (Fig. S3A–C, ESIw), and

analogous results were obtained with the synthetic peptides

(Fig. S3E and F, ESIw). Given these affinities and observations

indicating that TbRI-ED (residues 7–91) is more soluble than

TbRI-ED (residues 1–101), we employed the former in all

subsequent experiments.

Although TGF-b directly contacts both TbRI and TbRII in

the oligomeric complex, the TGF-b binding site on each

receptor is quite distinct.24,39 Given that Pep1 and Pep2

interact with both receptors and compete with each other for

binding, it seems unlikely they occupy the same regions as

TGF-b. Consistent with this analysis is the observation that

phage binding to TbRI and TbRII is unaffected by the

addition of TGF-b (data not shown). We used surface

plasmon resonance (SPR) to further explore peptide versus

growth factor binding. Specifically, TbRI-ED and TbRII-ED

were immobilized onto the sensor chip to test for competition

of the peptides with TGF-b. It is known that TGF-b alone has

weak affinity for the TbRI-ED.24 Consistent with our expecta-

tions, TGF-b1 did not bind detectably to the TbRI-ED surface

(Fig. 3B). In contrast, the TGF-b interaction with the TbRII-ED

surface was readily monitored. From the observed dose-

dependent SPR responses, an apparent Kd value of approxi-

mately 1.4 nM was determined (Fig. 3A). Previous SPR

studies have found that TGF-b1 binds to the monomeric

TbRII-ED with a Kd value of B100 nM,45 while it binds to

artificially dimerized TbRII-ED with Kd of B5 pM.46

The intermediate dissociation constant for TGF-b with

our TbRII-ED surface indicates that the surface presents

the receptor as a mixture of monomeric and dimeric forms.

Notably, when Pep1 was added as a potential competitor,

no significant changes in TGF-b binding to TbRII-ED

were observed (Fig. 3B). These results suggest that Pep1

and Pep2 share a previously unknown binding site on

TbRII.

As stated earlier, a direct binding assay cannot be used to

ascertain whether the peptide ligands compete with TGF-b for

binding to TbRI-ED. We therefore employed a cell-based

functional assay. If the peptide ligands occupy the TGF-bbinding site on either receptor, TGF-b1-regulated gene

expression should be affected. This possibility was evaluated

using a mink lung epithelial cell line (Mv1Lu) stably trans-

fected with a TGF-b-responsive reporter gene. The gene

construct consists of twelve repeats of a Smad binding element

(SBE) with a sequence of CAGA (SBE(CAGA)12) immediately

upstream of the luciferase sequence.36 When the transfected

cells are treated with TGF-b1, Smad3 translocates into the

nucleus and binds to the SBE(CAGA)12 sequence thereby

promoting the expression of a gene encoding luciferase.

The production of luciferase is readily quantified. TGF-b1regulated luciferase gene expression with an EC50 value of

approximately 10 pM (Fig. S4A, ESIw). As expected, the

addition of 10 mM TbRI kinase inhibitor SB-431542

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completely blocked the TGF-b-induced luciferase gene

expression (Fig. 3C), demonstrating TGF-b indeed func-

tions through TbRI. To test whether Pep1 and Pep2 compete

with TGF-b1, a titration with each peptide ligand was

conducted with 10 pM of TGF-b1. Neither Pep1 (Fig. 3C)

nor Pep2 (Fig. 3D) affects TGF-b1-regulated luciferase gene

expression. Additionally, the peptides alone had no effect on

the baseline luciferase gene expression (Fig. S4B, ESIw).47

Together, our results demonstrate that Pep1 and Pep2 occupy

the same binding sites on both TbRI-ED and TbRII-ED, and

these sites are distinct from those used by the natural growth

factor.

These findings indicate that our phage panning experiment

has identified hot spots for ligand interactions within TbRI

and TbRII. Previous studies using phage display have sug-

gested that natural protein-binding sites have intrinsic proper-

ties that predispose them to ligand binding.27,28,48,49 It is

therefore likely that the sites identified by our phage-derived

peptide ligands are used by endogenous proteins.

Dendrimers as platforms to display multiple copies of Pep1

The observed difference (105-fold) in binding affinities between

monovalent synthetic peptides and that of the phage particles

(bearing 3–5 copies of the peptides) suggests that multivalent

ligands for TbRI and TbRII will be more potent. Multivalent

binding is an intrinsic feature of TGF-b receptor signaling, as

the active complex involves 2 copies of each receptor and both

TbRI and TbRII form dimers or oligomers on the plasma

membrane of the cell surface.18 Thus, we postulated that the

functional affinities of the peptides could be increased by

multivalent display50,51 and that multivalent ligands would

serve as valuable probes. To this end, we employed generation

3 PAMAM dendrimer38 as a scaffold for peptide attachment

(Fig. 4A). This framework was chosen because the dendrimer

is extremely water-soluble and possesses many (a maximum of

32) primary amino groups as potential peptide conjugation

sites. Its high molecular weight also is valuable because its

binding can be detected readily by using SPR.52,53 The strategy

Fig. 3 Pep1 and Pep2 do not compete with TGF-b in binding to either TbRI-ED or TbRII-ED. (A) Binding of TGF-b1 (41 pM to 30 nM) to

TbRII-ED was tested using SPR. TbRI-ED and TbRII-ED were immobilized through their lysine residues. A protein-free flow cell was used as

control. TGF-b binds to TbRII-ED with a saturating concentration of 10 nM. At the concentrations tested, TGF-b1 has no observable affinity to

TbRI-ED (data not shown). (B) Pep1 does not compete with TGF-b in binding to TbRII-ED. (C) TGF-b1 initiated luciferase gene expression in an

Mv1Lu reporter cell line stably transfected with a SBE(CAGA)12-luciferase reporter gene. TbRI kinase inhibitor SB-431542 inhibited TGF-bregulated luciferase gene expression. Pep1 and (D) Pep2 do not alter the cellular response to TGF-b1 (10 pM).

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for functionalizing the dendrimer involved mimicking the

presentation of the peptide on phage, which is displayed as a

fusion to the N-terminus of the PIII coat protein. Accordingly,

we appended the C-terminus of Pep1 to the dendrimer. Con-

jugation was mediated through a PEG8 crosslinker which

contains a succinimidyl ester at one end and a maleimide at

the other. While the linker contains two electrophilic groups,

the dendrimer amino groups react preferentially with the

succinimidyl ester moieties. Pep1 was subsequently coupled

to the maleimide-displaying dendrimer through conjugate

addition of the C-terminal cysteine residue (Fig. 4B). The cysteine

thiolate is an excellent nucleophile that can undergo selective

and rapid conjugation to the maleimide.54 The resulting

dendrimer has a molecular weight of B35 kDa based on the

SDS-PAGE and MALDI mass spectrometry analysis, indicating

that it bears approximately 5 peptide moieties.

The avidity of Pep1-presenting dendrimers for the TbRI and

TbRII was evaluated using SPR. The dendrimer was injected

over TbRI-ED- and TbRII-ED-functionalized flow cells, as

well as an ethanolamine-functionalized control. Binding of the

dendrimer was detected when it was used at nanomolar

concentrations (KdE 10�7 M), indicating that it is an excellent

ligand (Fig. 5A and B). This dendrimer binds to TbRI-ED and

TbRII-ED with similar affinities, consistent with phage ELISA

results. This observation is intriguing. Indeed, although type I

and type II receptors in the TGF-b superfamily are distinct by

sequence comparison, they are structurally related. Specifi-

cally, they have a common pattern of four disulfide bonds,

stabilizing a structure feature named the ‘‘three-finger toxin

fold’’24,55 (Fig. 1B). This structural feature also is shared with

other TGF-b superfamily members, including the bone morpho-

genic protein receptor IA (BMPR-IA) and the activin receptor II

(ActRII).25 These observations raised the possibility that

our peptide ligands recognize other receptors in the TGF-bsuperfamily.

To test whether binding of the dendrimer is specific for

TbRI-ED and TbRII-ED, we assessed its affinity for two of the

aforementioned TGF-b family members: BMPR-IA and

ActRII. The extracellular domain of BMPR-IA (BMPR-IA-ED)

and ActRII (ActRII-ED) were immobilized on the SPR sensor

chip. The activity of these immobilized receptors was verified

by their ability to bind BMP-4, a known ligand56 (Fig. S5, ESIw).Interestingly, even at high dendrimer concentrations (6 mM),

no interaction of the dendrimer with BMPR-IA-ED nor

ActRII-ED could be detected (Fig. 5C and D). These results

demonstrate that Pep1 interacts specifically with TbRI-ED

and TbRII-ED but not with the closely related BMPR-IA or

ActRII.

In addition to receptors closely related to TbRI and II, we

also tested if our dendrimer binds to b-glycan and endoglin.

These proteins were chosen as controls for two reasons. First,

although they are coreceptors for TGF-b signaling, b-glycanand endoglin are not related to TbRI and TbRII; therefore, a

peptide ligand for the receptors should not show any affinity to

these receptor ligands. Secondly, b-glycan and endoglin are

members of a large class of proteoglycans that are modified

with heparan sulfate- or condroitin sulfate-containing glyco-

saminoglycans (GAGs). Anionic GAGs are abundant at the

plasma membrane of eukaryotic cells and in the extracellular

matrix; a specific ligand for TbRI and TbRII should not

interact with these species. The extracellular domain of endoglin

(endoglin-ED) and b-glycan-ED were immobilized on the SPR

sensor chip. As expected, no interaction of the dendrimer

with endoglin-ED or b-glycan-ED could be detected at any

Fig. 4 Multivalent display of Pep1 on G3 dendrimer. (A) Peptides identified from phage display can be displayed on multivalent scaffolds to

afford ligands with increased avidity. (B) Synthetic scheme for conjugating Pep1 to G3 PAMAM dendrimer.

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concentration tested (Fig. 5E and F). These results indicate that

Pep1 binds to TbRI-ED and TbRII-ED specifically. Thus, the

multivalent display of Pep1 can increase its functional affinity

by approximately 100-fold while retaining high specificity. This

finding is consistent with our previous observations indicating

that a small change in ligand affinity for different targets can be

amplified when a ligand is displayed multivalently.57 As a result,

multivalent ligands can show enhanced functional affinity and

specificity.57

We anticipate that dendrimer display can serve as a general

strategy to facilitate the characterization of low affinity peptide

ligands. Peptide hits from a first generation phage library

screening can have relatively weak affinities (e.g. 10�4 M),

which complicates characterizing their relative affinities and

specificities. Indeed, peptide characterization is often the

rate-limiting step in ligand optimization. False positives, as

well as false negatives, can arise that undermine the design of

effective second generation libraries. Dendrimer-displaying

peptides can overcome this limitation because their increased

affinity and molecular weight render them useful probes in

SPR assays. The peptide-substituted dendrimers provide other

attractive features such as their size and the opportunities they

present for introducing multifunctionality. For example, steric

effects from dendrimer binding might result in an increase in

its potency.50 In addition, because a dendrimer molecule can

display many sites for functionalization, a label such as a

fluorophore or a nanoparticle can also be appended.53 Such a

label could facilitate the characterization of the peptide

Fig. 5 (A) Binding affinities of the dendrimer to TbRI-ED, (B) TbRII-ED, (C) BMPR-IA-ED, (D) ActRII-ED, (E) endoglin-ED and (F)

b-glycan-ED were assessed by SPR. All proteins were immobilized through their lysine residues. A protein-free flow cell was used as control. The

dendrimer binds to TbRI-ED and TbRII-ED, but not to endoglin-ED at concentrations ranging from 2.93 nM to 1.5 mM. In a separate

experiment, the dendrimer did not interact with BMPR-IA-ED, ActRII-ED or b-glycan-ED at concentrations ranging from 47 nM to 6 mM.

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ligands, as well as their target. For example, such a conjugate

could be used to visualize58 or manipulate51 the targeted

protein on a cell surface. We note that dendrimeric probes

like the ones we describe that do not directly compete with the

growth factor ligand might be especially useful for probing

signaling and endocytosis.

Conclusions

In summary, we have used phage display to uncover peptide

ligands for the TbR-EDs. Although our screen focused on the

TbRI-ED, the peptides we found also bind to TbRII-ED with

similar affinities. To facilitate the characterization of the

peptide ligands, we displayed Pep1 on a dendrimer scaffold

to afford a ligand with excellent functional affinity. The

resulting dendrimer interacts with TbRI-ED and TbRII-ED,

but not with related receptors. This finding suggests that

there are intrinsic ligand-binding hot spots on TbRI-ED and

TbRII-ED uncovered by phage panning. These sites are

distinct from those occupied upon TGF-b binding, suggesting

that the peptide ligands target novel binding sites. Based on

the hot spot theory in protein–protein interactions,27,48 it is

likely that these newly identified binding sites are exploited by

endogenous proteins. Specifically, they may be used by

coreceptors that enhance or modulate TGF-b signaling. Given

the importance of cell-surface receptor oligomerization in

TGF-b signaling, the identification of peptides that bind to

both TbRI and TbRII suggest that multivalent ligands might

be used to control TGF-b signaling.59

Acknowledgements

This research was supported by the University of

Wisconsin, Materials Research Science and Engineering

Center (DMR-0520527, to LL), NIAID (AI055258, to LLK),

NIH (GM58670, to APH) and the Robert A. Welch Founda-

tion (AQ1431, to APH). We thank Dr Eric S. Underbakke,

Adam H. Courtney and Dr F. Michael Hoffmann for helpful

discussions on phage display and TGF-b signaling. We thank

Dr Gary L. Case for help with automated peptide synthesis

and Dr Matthew R. Levengood for help with MALDI analysis.

SPR data were obtained at the University of Wisconsin-

Madison Biophysics Instrumentation Facility (BIF). We

thank Dr Darrell R. McCaslin for helpful conversations on

SPR experiments.

Notes and references

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